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AND BIOENGINEERING 1999
Effect of Soybean Oil on Oxygen Transfer in the Production of Tetracycline with an Airlift Bioreactor
SHIRU JIA,’ GUIBIN CHEN,’ PRIHARDI KAHAR,2 DU BOK CHOL3
Department of Biochemical Engineering, Tianjin University of Light Industry, 300222 Tianjin, China,’ Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan,2 and Institute of Life Science, Chosun University, 375 Seo-Suk Dong, Dong-Gu, Kwang-Ju, South Korea3
Received 14 January 1999/Accepted 15 March 1999
Corn starch and soybean oil are suitable carbon sources for the production of tetracycline by Streptomyces aureofacience CG-1. However, it could not produce more than 6 g/l of tetracycline even if initial corn starch concentration was increased to more than 100 g/Z. It was confirmed by shaking flask experiments that the kLa in a mixture of 296 soybean oil in water was four folds compared with that without soybean oil. With the addition of soybean oil to the starch medium in a shaking flask, tetracycline production was significantly improved. By scaling-up to a 55Zairiift bioreactor from SOO-ml Erlenmeyer flask, more than 10 gNof tetracycline was produced with the addition of 60 g/Z of soybean oil to the medium contsining 100 g/Z of corn starch. The dissolved oxygen level in the airlift bioreactor containing soybean oil was higher than that without soybean oil. This suggeststhat soybean oil is not only a suitable carbon source but is also a surface-active agent which may accelerate the oxygen transfer. This may lead to the possibility of the enhanced production of tetracycline at a low cost in airlift bioreactor. [Key words: tetracycline, soybean oil, Streptomycesaureofaciens, airlift bioreactor]
Tetracycline (TC), which is a broad-spectrum antibiotic, can inhibit the growth of gram-positive and gramnegative bacteria, some kinds of Rickettsia, viruses and protozoa. Moreover, TC can be used as an intermediate and synthesised into some excellent semi-synthetic derivatives by modifying the chemical structure. Therefore, TC has a vast scope of use. We are attempting to confirm the possibility of enhancing the production of TC using an airlift bioreactor, because airlift bioreactors have many economical implications in regard to reactor construction, maintenance and scaling-up as previously reported (1, 2). However, the ability of airlift bioreactors to supply oxygen is generally lower than that of conventional type of bioreactors, i.e., aeration and stirred tank bioreactors. Therefore, it is necessary to determine the optimal culture conditions suitable for the airlift bioreactor as previously reported. In this study, the medium and culture conditions were designed so that TC may be produced efficiently at low oxygen supply conditions in a 500-ml Erlenmeyer flask, followed by scaling-up to 5.5-l airlift bioreactor (3). The TC-producing strain used in this study was Streptomyces aureofaciens CC-l. For production of TC, the following media were used. (a) Slant medium (g/l distilled water): bran, 36; MgS04.7H20, 0.1; KH2P04, 0.3; agar, 20; (b) seed medium (g/l tap water): soybean meal, 20; corn starch, 40; peptone, 5; yeast extract, 5; CaC03, 4; (NH4)2S04, 3; NaBr, 2; MgS04. 7H20, 0.25; KH2P04, 0.2; and (c) production medium (g/l tap water): soybean meal, 30; corn starch, 100; yeast extract, 2.5; peptone, 15; CaC03, 7; (NH4)2S04, 2.5; NaBr, 1.5; MgS04.7H20, 0.25. These media were sterilized at 121°C for 30min. In the case of using corn starch as a carbon source, the corn starch was partially hydrolyzed by the addition of 0.2% cY-amylase (HS, NAGASE Biochem. Ind. Ltd.,
* Corresponding author.
Kyoto) at 95°C for 40 min. One loopful of Streptomyces aureofaciens CC-1 was transferred to the slant medium and cultured at 31°C for 5 d. Then one loopful of the slant culture of Streptomyces aureofaciens was inoculated into a 500-ml Erlenmeyer flask containing 40ml of the seed medium and cultivated at 31°C for 17 h on a reciprocating shaker at 150rpm. For the production of TC, 10% of the seed culture was inoculated into a 500~ml Erlenmeyer flask containing 40ml of the production medium, and to a 5.5-l airlift bioreactor containing 4 I of the production medium. The schematic structure of the 5.5-l airlift bioreactor was previously reported (3). Cultivation in the Erlenmeyer flask was carried out under the above-mentioned conditions for 144 h. The airlift bioreactor was operated at 31°C for 144 h. The aeration rate during the cultivation in the airlift bioreactor was fixed at 2 vvm. Since this aeration rate was such that the working liquid volume could be decreased due to evaporation, the culture volume was maintained at 4 I by the addition of sterilised water to the reactor using a level sensor. The dissolved oxygen concentration in the airlift bioreactor was measured with a DO meter (Tianjin University of Light Industry, Tianjin, China). The TC concentration was determined by a spectrophotometric method (4). The concentrations of cells and the carbon sources were measured as described previously (5). The volumetric oxygen transfer coefficient, kLa was measured by the method of Linek and Vacek (6). In generally, corn starch is often used as the sole carbon source for the production of various kinds of antibiotics. Table 1 shows the effect of initial concentration of corn starch on TC production in a shaking 500-ml Erlenmeyer flask. TC production was the highest at an initial corn starch concentration of 100 g/l. At concentrations higher than lOOg/l of corn starch, the TC production was rapidly decreased, possibly due to limitation of
0' oJ L 0 24 48 72 % 120 Time (h) 144 96 120 144 Time (h) FIG. 1201 loo- (b) -70 -60 OJ 0. Figure 2 shows the time-course of TC production when 60 g/l of soybean oil was added to the production medium containing 100 g/l of corn starch (b) and without addition of soybean oil (a). However. added. however. Therefore.5 6.8 9. .1 7.36. kLa.1 6. - (a) (b) 0’ 0.9 20.d 0’ 0’ Time (h) Time (h) FIG. Effect of initial concentration of starch as the sole carbon source on the production of TC 0 0. However. soybean oil can significantly enhance kLa. 1.2 J.0 11. it is very difficult to increase TC production only by increasing the initial concentration of corn starch. it is very difficult to use them for the commercial production of TC. Time courses of production 12^ 1.2 28. Starch (g/f) Tetracycline (g/f) Yield from starch (%) TABLE 2. Although the reason why the kLa was decreased at concentrations higher than 20 g/l of soybean oil in this model experiment is not clear yet. In order to increase the TC production to higher than 6 g/l. 1. Effect of the addition of soybean oil to the production medium containing 100 g/l of corn starch on the production of TC 0 20 30 40 50 60 80 100 Soybean oil (g/l) Tetracycline (g/[) 6.2 8. Effect of addition of soybean oil on the oxygen transfer coefficient. 3.8.8 7. 1. 2.5 9.0 10. we also attempted to confirm whether soybean oil.01 0.0120= IO- of TC in a shaking flask without (a) and with the addition of 60 g/l of soybean oil (b). We have previously reported that addition of perfluorocarbon or n-dodecane efficiently improved the oxygen transfer rate coefficient. BIOSCI.4 Yield from soybean oil (%) .0 4. = 3 m FIG. since they are expensive.2 80 5.3 100 6. Therefore. can improve the oxygen transfer coefficient.0 10. As clearly shown in Fig. soybean oil was added to the production medium containing lOOg/l of corn starch in the shaking 500-ml Erlenmeyer flask. as well as the aforementioned in a water-air system in 500-ml shaking flask experiments.6 60 5.5 19. the addition of more than 60 g/l of soybean oil rather led to a decrease in the TC production.8 40 3.. thereby eventually increasing the yield of yeast (3).2 6.5 I-airlift bioreactor without (a) and with the addition of 60 g/i of soybean oil (b). BIOENG. 1. TC production was significantly increased to more than 10 g/l with the addition of 6Og/l of soybean oil. it was confirmed that soybean oil can increase the kLa if added at an appropriate concentration.1 120 5.0 7.2 22.826 TABLE JIA ET AL. the kLa value was the highest when 2Og/l of soybean oil was added to a 500-ml Erlenmeyer flask with water. In this model experiment. which is often used as a carbon source for the production of various kinds of antibiotics (7-9). Time-courses of production of TC in a 5. However. As clearly shown in Table 2. k~a in a 500-ml Erlenmeyer flask with water.5 9. it decreased rapidly when more that 20 g/Z of soybean oil 121 10.4 0 10 20 30 40 Soybean oil (g/O the oxygen supply as viscosity of the culture broth increased.
87. S... Saito. it was maintained at higher than 10% from the first 48 h to 96 h (b). R. suggesting that soybean oil might be a suitable carbon source for the growth of Streptomyces aureofaciens CG-1 and production of TC. when soybean oil was not added. 413-417 (1998). Jia.5-I airlift bioreactor by adding soybean oil as an enhancer of oxygen transfer and a carbon source. 82. S. 70. M. K.. Y. Yahiro. 19. Y. Oka. and Osoao. and Vacek. Takahama. Choi. J. Y. Assoc. Biotechnol. On the other hand. 45 g/l of soybean oil was efficiently consumed. S. S. Y. REFERENCES 1. and Okabe. 40.. Ferment. 82. Li. J.. 191-193 (19%). and Okabe. Eiki. 773779 (1994). 66. M. S. Ferment. Ferment.. Linek. I. T.2g/l).: Comparison of airlift and stirred tank reactors for itaconic acid production by Aspergillus terreus.. S. 86. Sahav. J. Yahiro.. S. Bioeng. resulting in a lower TC production (6. Bioeng. J. Technol. The final soybean oil concentration was less than 2Og/l.: Efficient tylosin production from Streptomyces fradia using rapeseed oil.. Thus.. Park. J.. 686 (1973).. D.. Bioeng.: Comparison of neomycin production from Streptomyca fradiae cultivation using soybean oil as the sole carbon source in an airlift bioreactor and a stirred-tank reactor. S. R. H.. Seriue. Bioeng. Appl. Ishida. B. As shown in Fig. Y. 1999 NOTES 827 As clearly from the figure. Tsunoda...5 f-airlift bioreactor.: Improvement of tylosin production from Streptomyces fradiae culture by decreasing the apparent viscosity in an airlift bioreactor. Tamnra. Park. J. although the DO concentration decreased rapidly with increasing cell concentration.VOL. S. the DO rapidly decreased to nearly zero at 24 h (a)... A. Jia. and Okabe. suggesting that the soybean oil was consumed as rapidly as the corn starch. Y.. and Takeda. T. we attempted then to reproduce the data in a 5. Microbial. Tamura.: J. N. 443-448 (1995). Biotechnol.. 619-621 (1997). 183-186 (1996). 559-565 (1988).. Park. V. S. B. Park. Bioeng. K. Y. . 835-965 (1977). H. and Okabe. Y. D. 79.. B. Biotechnol.... Gushima. Okabe. Anal. M. Since we can increase TC production to more than log/l by adding soybean oil in a shaking culture in a WO-ml Erlenmeyer flask. M.. TC production was higher in medium with soybean oil than in that without it (a).: Production inhibition and its removal on josamycin fermentation by Streptomyces narnbonensis var.. M.. 3. Ohta.. Momose. and Okabe.: Chemical engineering use of catalyzed sulfite oxidation kinetics for the determination of mass transfer coefficient by dynamic model moment analysis. H. V.. Park. a higher TC production of more than log/l obtained from the Erlenmeyer flask cultivation was successfully reproduced in the 5. 60. Choi.. Park. T.: Enhancement of cephamycin C production using soybean oil as the sole carbon source. resulting in a higher TC production more than log/l at 144 h. Ferment. M.. Ferment. Lett. K.. Chem.: Enhanced oxygen transfer in tower bioreactor on addition of liquid hydrocarbons. S.. josamyceticus.